Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host
Bartonella bacilliformis, the etiological agent of Carrions disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrions disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs r45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9(BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro. Author summaryB. bacilliformis is a bacterial pathogen that is transmitted between humans by phlebotomine sand flies. Bacteria often express sRNAs to fine-tune the production of proteins involved in a wide array of biological processes. We cultured B. bacilliformis in vitro under standard conditions then shifted the pathogen for a period of time to ten distinct environments, including multiple temperatures, pH levels, and infections of human blood and human vascular endothelial cells. After RNA-sequencing, a manual transcriptome search identified 160 putative sRNAs, including seven highly-conserved sRNAs and 153 novel potential sRNAs. We then characterized two of the novel sRNAs, BbgpI and BbsR9. BbgpI is a group I intron (ribozyme) that self-splices and disrupts an unannotated gene coding for a transfer RNA (tRNACCUArg). BbsR9 is an intergenic sRNA expressed under conditions that simulate the sand fly. We found that BbsR9 targets transcripts of the ftsH, nuoF, and gcvT genes. Furthermore, we determined the specific sRNA-mRNA interactions responsible for BbsR9 binding to its target mRNAs through in vitro mutagenesis and binding assays.